Field AS, Chamberlain NL, Tran D, Morey AL. Suggestions for HER-2/neu testing in breast
carcinoma, based on a comparison of immunohistochemistry and fluorescence
in situ hybridisation. Pathology.2001;33:278-282.PubMedGoogle Scholar

Context Testing for HER-2 oncogene in breast cancer has increased because of
its role as a prognostic and predictive factor. Some advocate gene testing
by fluorescence in situ hybridization (FISH) vs protein testing by immunohistochemistry
as the method which most accurately evaluates and predicts response to the
anti–HER-2 antibody, trastuzumab. However, critical examination of FISH
on a screening basis has yet to be performed.

Design, Setting, and Patients A quality control and quality assurance program for HER-2 testing by
FISH, which used tumor specimens from 2963 patients (median age, 56 years)
with breast cancer received from 135 hospitals and cancer centers in 29 states,
was performed at a reference laboratory from January 1, 1999, to May 15, 2003.
Every specimen evaluated by FISH was parallel tested with immunohistochemistry
tests.

Main Outcome Measures With FISH as the presumed standard testing method, the positive and
negative predictive values and sensitivity and specificity of immunohistochemistry
were calculated.

Results A total of 3260 clinical HER-2 tests by FISH were performed on 2963
serially referred breast cancer specimens. Of these, 2933 tests were successful
and 2913 breast cancer specimens had both FISH and immunohistochemistry results
available. With FISH as the standard testing method, the positive predictive
value of positive immunohistochemistry score (3+) was 91.6%, and the negative
predictive value of negative immunohistochemistry score (0 or 1+) was 97.2%.
The sensitivity of immunohistochemistry tests, including tumor sections with
scores of 2+ or 3+, was 92.6% and the specificity of immunohistochemistry
tests with scores of 3+ was 98.8%. The FISH test had a significantly higher
failure rate (5% vs 0.08%) and reagent cost ($140 vs $10), and longer testing
(36 hours vs 4 hours) and interpretation times (7 minutes vs 45 seconds) vs
immunohistochemistry tests.

Conclusions A testing algorithm for HER-2 determination is most efficient by using
immunohistochemistry as the method of choice, with FISH performed for cancers
with indeterminate results (2+ score). Successful quality control and quality
assurance programs are a prerequisite for such approaches.

The human epidermal growth factor receptor 2, HER-2 (HER-2/neu or c-erbB-2), oncoprotein is overexpressed
in 20% to 25% of invasive breast cancers.1 Previous
studies have supported the importance of HER-2 overexpression as an independent
prognostic marker of clinical outcome,1-5 as
a predictor of benefit from doxorubicin-based chemotherapy,6,7 and
for metastatic disease, for benefit from the anti–HER-2 antibody, trastuzumab.8-11 Determination
of HER-2 status is now an integral part of the clinical-pathological workup
of breast cancer.

The HER-2 protein overexpression is usually a direct consequence of
gene amplification.1,12 This unique
gene-protein relationship has spawned several methods for assessing HER-2
status. Currently, immunohistochemistry and fluorescence in situ hybridization
(FISH) are the most widely used, both because these technologies are ideally
suited for routine and archival paraffin-embedded tissue and are evaluated
by direct visualization of tumor cells. Published gene-protein correlation
studies in general show good-to-excellent concordance.13-26 Initial
studies evaluating which method best predicts clinical outcome have shown
better clinical correlation with FISH than with immunohistochemistry assays.15,27 Some clinical investigators have
therefore advocated the use of FISH as the method of choice for HER-2 testing.22,27,28 However, in many
of these assessments, immunohistochemistry assays were inadequately optimized,
rendering comparison between these differing methods of limited utility. This
is further compounded because immunohistochemistry is more widely used than
FISH in community laboratories, leading to inaccurate results.29,30 Thus,
based on such studies, it may be incorrect to conclude that FISH testing should
replace immunohistochemistry as the screening tool of choice in breast cancer
patients.

The use of FISH as a widely used screening assay has not been critically
evaluated. Some of its documented disadvantages include high reagent cost
and longer procedure and interpretation time.13 We
examined the relationship between FISH and immunohistochemistry testing for HER-2, using tests
approved by the Food and Drug Administration (FDA), in a large number of breast
tumor samples and recommend a practical testing algorithm to screen for HER-2
in breast cancer.

Methods

Patients and Study Design

From January 1, 1999, to May 15, 2003, a quality control and quality
assurance program for HER-2 testing was commenced at PhenoPath Laboratories,
Seattle, Wash. For every FISH assay requested by a referring physician, an
accompanying immunohistochemistry test was set up in parallel. A total of
2963 patients with breast cancer were accrued during this interval. Patients'
ages ranged from 28 to 87 years, with a median age of 56 years. Tumor sections
were received from 135 hospitals and cancer centers in 29 states. The reason
for requesting FISH testing by the referring physicians was to determine HER-2/neu gene status on tumor sections with indeterminate (2+
score) immunohistochemistry results (52%), to confirm gene amplification on
tumor sections with positive (3+ score) immunohistochemistry results (24%),
and to verify gene status on tumor sections with negative (0 or 1+ score)
immunohistochemistry results (23%). The University of Washington Human Subjects
Division has determined that this research activity qualifies as exempt from
US federal regulations.31

Tissue sections were deparaffinized and rehydrated before incubating
them in 0.01-M citrate buffer at pH 6.0 in a steamer for 40 minutes at more
than 95°C. A polyclonal antibody to HER-2/neu (A0485,
DAKOCytomation, Carpinteria, Calif) was applied to sections and incubated
for 40 minutes at room temperature. With intervening wash steps in phosphate-buffered
saline, slides were incubated for 30 minutes at room temperature in a rabbit-specific
labeled polymer (DAKOCytomation, Carpinteria, Calif), followed up by 10 minutes
at 37°C in a solution containing 3% hydrogen peroxide and 3,3′-diaminobenzidine.
Slides were counterstained with hematoxylin. Immunostained slides were scored
according to a modification of the scoring system approved by the FDA, as
described previously.32 In brief, because of
the multitude of sources of specimens with a wide range of fixatives and processing
techniques, we developed a "subtraction scoring" method, which counts any
visible signal of the nonneoplastic breast epithelium as negative and subtracts
the score of the tumor cells from that of the benign cells.32 When
the subtraction score is 2+, we used the term indeterminate, given the uncertainty about predictability of FISH results among tumor
sections with a score of 2+. Such results are not to be confused with a failed
test, which was defined in this study as failure to produce interpretable
results after an additional testing attempt.

The FISH studies to assess HER-2 gene status were performed by using
reagents (Vysis Inc, Downers Grove, Ill), according to the manufacturer's
guidelines. Pathologists (H.Y., A.M.G., L.C.G., T.S.B., R.W., H.H.) scored
the FISH studies, counting at least 60 tumor cells. Results were reported
as a ratio of HER-2 to chromosome 17 signals.

Quality Control and Quality Assurance Programs

Digital images of FISH and immunohistochemistry studies were stored
and subsequently used for quality control conferences. For every batch of
tumor sections, appropriate quality control sections were used for FISH testing.
Sections were declared not interpretable when it was impossible to accurately
determine the number of gene or chromosome copies on the tumor cells, even
with adequate quality control sections. All FISH tests on these sections were
repeated once before results were considered inconclusive. Quality control
sections for immunohistochemistry tests included individual 3+ positive tumor
sections, multiple cell pellet sections with known ranges of protein expression,
and gene status. Each FISH test was simultaneously accompanied by a hematoxylin
and eosin-stained section and immunohistochemistry section. In the same order
with each case, the pathologist examined the hematoxylin and eosin-stained
section and scored the immunohistochemistry results before scoring the FISH
test. Following the determination of FISH status, pathologists reviewed the
immunohistochemistry-stained section on the same case without modifying the
score, regardless of any immunohistochemistry-FISH discrepancy. Results of
the immunohistochemistry and FISH scores were entered in a computerized database
(Fourth Dimension Inc, San Jose, Calif), transferred to Microsoft Excel spreadsheet
(Microsoft Corp, Redmond, Wash) after each test batch, and then exported to
JMP version 5.0 statistical software (SAS Institute Inc, Cary, NC).

A number of retrospective analyses were conducted on a regular basis.
In addition to a monthly review of small random batches of tumor sections
(10-40), the retrospective analysis also included targeted reviews of all
immunohistochemistry false-negative and false-positive tumor sections and
possible sources of discrepancy were identified and arranged for future reviews.
The quality control and quality assurance program also included periodic blinded
review of the immunohistochemistry-stained sections to the FISH results, with
intraobserver and interobserver comparisons.

Results

A total of 3260 clinical HER-2 tests by FISH were performed on 2963
specimens from patients with breast cancer tumors. Of these, 2933 tests were
successful and 2913 breast cancer specimens had both FISH and immunohistochemistry
test results available. The FISH failure rate was 5% (n = 163), and the immunohistochemistry
failure rate was 0.08% (n = 25) after a second and final test attempt. Table 1 shows the relationship between
FISH and immunohistochemistry test results.

When FISH was presumed to be the standard testing method, the positive
predictive value of positive immunohistochemistry scores (3+) was 91.6% and
the negative predictive value of a negative immunohistochemistry score (0
or 1+) was 97.2%. We also calculated the sensitivity and specificity of immunohistochemistry.
These analyses were performed with and without the tumor group that had indeterminate
(2+ score) results (Table 2).

The mean direct reagent cost by vendors of each FISH and immunohistochemistry
test was $140 and $10, respectively. The mean (SD) procedure time was 36 hours
(30 minutes) for FISH and 4 hours (12 minutes) for immunohistochemistry. Mean
(SD) time for the pathologist to interpret the test was 7 minutes (2.5 minutes)
for FISH and 45 seconds (13 seconds) for immunohistochemistry (Table 3).33

The concordance rate between immunohistochemistry and FISH tests was
64.9% when scores of 2+ and 3+ positive tumor sections were grouped together.
However, when the 2+ score group (1151 tumor sections) was excluded from the
analysis, the concordance rate improved to 96.1%. This number is very high
because a large subset of tumor sections (52.0%) was initially submitted for
FISH testing as a result of an indeterminate score (2+) by immunohistochemistry
by the referring pathologists. Among the FISH-negative tumor sections (n =
2371), only 1.2% (n = 28) had an immunohistochemistry score of 3+, and the
remaining tumor sections were either negative (58.5%, n = 1388) or indeterminate
(40.3%, n = 955).

When FISH-positive results were divided into low and high levels of
gene amplification (using a gene/chromosome ratio cutoff of 5), only 2.8%
of the high-level group had negative immunohistochemistry results and the
majority of these tumor sections had immunohistochemistry scores of either
3+ (77.9%) or 2+ (19.3%). In the low-level group (gene/chromosome ratio of
2 or more but less than 5), 32.7% had an immunohistochemistry score of 3+
and the remaining test results showed predominantly (54.9%) a score of 2+,
with 12.5% negative immunohistochemistry results.

Using a set of 117 tumor sections to test the variability among pathologists,
complete agreement by 5 pathologists (H.Y., L.C.G., A.M.G., T.S.B., H.H.)
was reached on 50.4% of the sections, with 4 of 5 pathologists reaching an
agreement on an additional 24.7% of the sections. No 2-step discrepancy in
immunohistochemistry scoring (ie, 3+ score vs 1+ or 0 score) was observed
in any of the sections.

Comment

Testing methods for HER-2 are currently heatedly debated among pathologists
and oncologists involved in breast cancer, with no agreed-upon standard practices.
The Center for Biologics Evaluation and Research at the FDA has recognized
the limitations of all current HER-2 testing methods, stating that it "views
both immunohistochemistry and FISH as semi-quantitative if performed under
ideal circumstances. Both methods require subjective interpretation."34 Although pathologists commonly perform first-line
screening by immunohistochemistry with confirmatory FISH testing for indeterminate
immunohistochemistry test results, trastuzumab clinical trials have reported
better use of FISH in predicting treatment response, suggesting that FISH
should be used as a primary screening test.9-11 However,
when positive immunohistochemistry test results (3+ score) were analyzed separately
from indeterminate test results (2+ score) in the same trials, immunohistochemistry
was predictive of response.10,34,35

Moreover, the critical appraisal of FISH as a screening test for HER-2
has not been accomplished. A screening test to be routinely performed on a
large number of clinical specimens should be easy to perform and interpret,
show high rates of sensitivity, exhibit high levels of interlaboratory and
intralaboratory reproducibility, and be relatively inexpensive. The FISH method
for HER-2 testing should be critically assessed against these criteria before
implementing FISH as the first-line screening tool for HER-2 in breast cancer.

Trastuzumab acts at the protein and not at the gene level. Although
protein levels directly reflect the gene status and thus can be indirectly
measured via FISH method, emerging data show response to trastuzumab in patients
with positive immunohistochemistry score whether or not gene amplification
is present, arguing for the existence of an alternative mechanism of protein
overexpression other than gene amplification.10,34,35 Protein
expression as a predictor for clinical response independent of the gene status
supports a true biological basis for the discrepancy between the 2 methods.
Additionally, patients whose test results show chromosome 17 polysomy without
gene amplification (increased gene copy number secondary to aneuploidy of
chromosome 17) are currently not judged to be eligible for trastuzumab by
the FISH method because their test results are scored as negative. In our
study, this subset of tumor sections is quite large, approaching one third
(29.6%, n = 870; data not shown) of the entire cohort comparable with what
was previously reported (24.8%) in another large study.27 Determination
of response rate to trastuzumab for this group of patients is crucial given
the large size of this cohort. Widely performed testing with HER-2 FISH assays
that do not contain a probe for chromosome 17 report positive HER-2/neu gene status for tumor sections with polysomy of chromosome
17 and single copy HER-2/neu gene.

Another area of concern is the uncertain clinical significance of low-level
gene amplification. To our knowledge, no clinical trials have reported the
relationship between low-level amplification and response to trastuzumab.8-11 It
is possible that this category of patients may well be clinically classified
as indeterminate for amplification. Until data evaluating low-level amplification
become available, all otherwise eligible patients whose test results are within
this range will continue to receive trastuzumab without a critical assessment
of their likely response to treatment. In our study, the fraction of patients
with low-level gene amplification constitutes 47.4% of the positive tumor
sections (Table 1).

Preliminary findings from 2 cooperative group trials testing the adjuvant
use of trastuzumab, National Surgical Adjuvant Breast and Bowel Project B31
and North Central Cancer Treatment Group N9831, showed high rates of discordance
in HER-2 immunohistochemistry testing between community practices and a central
laboratory.29,30 When comparing
FISH testing between laboratories, B31 and N9831 preliminary assessment showed
5% and 23% disagreement, respectively, on FISH results (positive vs negative)
between 2 central laboratories.29,30 Additionally,
repeat FISH testing on a large subset (n = 198) of breast cancers by a central
laboratory showed 16% disagreement with previously reported results by another
central laboratory.34

The current study demonstrates the very tight correlation between immunohistochemistry
and FISH as part of a quality control and quality assurance program. Negative
immunohistochemistry test results in our study accurately predicted FISH results
in 97.2% of tumor sections, with a false-negative rate of 2.8%. The reasons
for the very low rate of false-negative immunohistochemistry results are multifactorial.
First, technologists and pathologists at the same institution are performing
the technical and interpretation aspects of the FISH and immunohistochemistry
tests. Also, FISH testing has improved the quality control and quality assurance
of HER-2 testing by immunohistochemistry at this institution. In the past,
our main immunohistochemistry monitoring method and scoring approach was periodic
verification of the percentage of HER-2 positive breast cancers. The FISH
test now provides another "fine-tuning" tool for evaluating HER-2 testing
by immunohistochemistry. Finally, performing side-by-side parallel immunohistochemistry
testing for all tumor sections tested by FISH has been a powerful quality
control mechanism providing instant and continuous feedback on the varied
technical and scoring parameters associated with both of these technologies.
Similarly, some of the reported high rates of false-positive and false-negative
immunohistochemistry results are secondary to multiple factors. Technically,
lack of adherence to strict quality control measures in accepting or rejecting
a test result, absence of internal quality control programs that identify
overscoring or underscoring of immunohistochemistry results are contributing
factors. At times, the type of specimen fixation (particularly alcohol or
alcoholic formalin) could significantly increase the sensitivity of the method
so tumor sections with negative (0 or 1+ signal) or indeterminate (2+) scores
may show stronger signal intensity. If the subtraction score was not performed
on such tumor sections, they may inadvertently be scored as positive (3+)
for overexpression.

Economic and practical considerations would tend to favor immunohistochemistry
over FISH as a screening tool, because these are very important given the
high incidence of breast cancer. The American Cancer Society predicted 211 300
new cases of invasive breast cancer in the United States during 2003.33 Given the known incidence of HER-2 alterations in
breast cancer, 70% to 75% of tumor sections tested by immunohistochemistry
will be negative (0 or 1+ score), and the remainder divided almost equally
between indeterminate results (2+ score) and positive (3+ score) overexpression.
Therefore, only about 15% of patient samples that are screened primarily by
immunohistochemistry would be indeterminate and require confirmatory FISH
testing. In the United States, if FISH testing were adopted as the standard
for all cases, we conservatively estimate the additional health care cost
would range from $50 to $70 million in 2003, for supplies and reagents alone.
The additional time allocated for FISH testing and scoring by pathologists
is also disproportionately high compared with immunohistochemistry. At our
institution, pathologists dedicate a substantial amount of time for scoring
and reporting of FISH tests compared with immunohistochemistry (Table 3).

Screening by immunohistochemistry and confirming indeterminate results
with FISH testing has been advocated by many investigators,14,15,18,20,36,37 with
some suggesting confirmation of both indeterminate and positive immunohistochemistry
results with FISH.26 Other studies favor FISH
testing as the primary screening test.22,27,28,38 An
international panel from 10 western countries has recently recommended immunohistochemistry
to be the screening method of choice, with repeat FISH testing on the indeterminate
subset. This panel also emphasized strict quality control and quality assurance
measures for any given technology.37 The results
in our study support these measures and recommend a standard procedure for
any laboratory that provides clinicians with either test. Lack of such measures
may explain the suboptimal reproducibility of the results of FISH tests performed
in community settings and repeated by the central laboratories of the cooperative
group trials.29,30 In fact, after
amending the B31 protocol to require entry HER-2 testing from laboratories
approved by the National Surgical Adjuvant Breast and Bowel Project (based
on volume of testing and documented immunohistochemistry/FISH concordance),
the false-positive rate has decreased to 3%.39 In
the case of HER-2 testing, once the results of such programs are translated
into successfully high rates of immunohistochemistry/FISH concordance, immunohistochemistry
can be reliably offered as a first-line screening test requiring confirmation
by FISH only in a small subset of cases that show indeterminate results (Figure 1).

In conclusion, accurate testing for HER-2 in breast cancer can be achieved
using immunohistochemistry with complementary FISH testing, taking advantage
of the strength of each of these technologies. Such an algorithmic approach
is especially important given the increasing number of prognostic and predictive
biomarkers in oncology.40

Field AS, Chamberlain NL, Tran D, Morey AL. Suggestions for HER-2/neu testing in breast
carcinoma, based on a comparison of immunohistochemistry and fluorescence
in situ hybridisation. Pathology.2001;33:278-282.PubMedGoogle Scholar